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. 2013 Jun 19;24(6):1057-67.
doi: 10.1021/bc400122c. Epub 2013 May 30.

Enzyme-mediated methodology for the site-specific radiolabeling of antibodies based on catalyst-free click chemistry

Brian M Zeglis  1 , Charles B Davis, Robert Aggeler, Hee Chol Kang, Aimei Chen, Brian J Agnew, Jason S Lewis
Affiliations

Enzyme-mediated methodology for the site-specific radiolabeling of antibodies based on catalyst-free click chemistry

Brian M Zeglis et al. Bioconjug Chem. .

Abstract

An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal (89)Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with (89)Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing (89)Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to highly selective tumor uptake (67.5 ± 5.0%ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic.

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Figures

Figure 1
Figure 1
SDS-PAGE of unmodified (lanes 1, 2), GalNAz-modified (lanes 3, 5), or DIBO-DFO-modified (lanes 4, 6) J591 antibody constructs either untreated (lanes 1, 3, 4) or treated (lanes 2, 5, 6) with PNGaseF. The black and white arrows indicate the antibody heavy chain and light chain, respectively, and the bands at 36.5 kDa are the result of excess PNGaseF enzyme.
Figure 2
Figure 2
Coronal PET images of 89Zr-DFO-DIBO/GalNAz-J591 and 89Zr-DFO-NCS-J591 (11.1 – 12.9 MBq [300–345 μCi] injected via tail vein in 200 μL 0.9% sterile saline) in athymic nude mice bearing subcutaneous, PSMA-expressing LNCaP prostate cancer xenografts (white arrows) between 24 and 120 h post-injection.
Scheme 1
Scheme 1
(A) Structures of UDP-GalNAz and DIBO-DFO; (B) Strain-promoted, catalyst-free click ligation between UDP-GalNAz and DIBO-DFO. DIBO = dibenzocyclooctyne; UDP = uridine-5′-diphosphate; GalNAz = N-azidozacetylgalactosamine; DFO = desferrioxamine.
Scheme 2
Scheme 2
Four step strategy for the site-selective, enzyme- and click chemistry-mediated radiolabeling of antibodies on the heavy chain glycans.
See this image and copyright information in PMC

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